全局转录调控因子CodY在嗜热链球菌中的调控机制研究
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摘要
嗜热链球菌(Streptococcus thermophilus)是重要的工业菌种,与德氏乳杆菌保加利亚亚种(Lactobacillus delbrueckii subsp. bulgaricus)或者瑞士乳杆菌(Lactobacillus helveticus)混合发酵生产酸奶和硬奶酪。嗜热链球菌年消费量达1021菌体细胞,是公认的食品级安全性菌株(Generally Recognized as Safe, GRAS)。长期生长于牛乳环境,嗜热链球菌形成了独特的乳糖转运和代谢系统,使其能够高效利用牛乳中的乳糖为菌体生长提供能量。由于嗜热链球菌的胞外蛋白酶活性较弱或者缺失,不能有效利用牛乳中的酪蛋白；同时牛乳中游离氨基酸和寡肽浓度低,限制了其生长。为了更好的适应牛乳环境,嗜热链球菌通过基因水平转移(Horizontal Gene Transfer, HGT)获得了多种氨基酸合成途径。此外,嗜热链球菌在生长和发酵过程中常常受到氧、温度和噬菌体等胁迫压力,影响菌体的生长和存活。为了能够充分利用环境中的营养物质和抵抗胁迫环境,细菌通过全局转录调控因子对胞内基因进行转录控制,形成一个庞大的代谢调控网络。全局转录调控因子CodY作为一个独特的转录调控蛋白普遍存在于革兰氏阳性菌,在细菌代谢、环境适应和致病性控制等方面都具有重要的调控作用。CodY通过与调控基因的启动子区域结合控制结构基因的转录,而能量信号分子鸟嘌呤三磷酸(Guanosine-5'-Triphosphate, GTP)和营养信号分子分支氨基酸(Branched Chain Amino Acid, BCAA)能够有效促进CodY和调控基因的作用,但是不同菌株CodY的作用机制不同。嗜热链球菌碳、氮代谢途径研究相对清楚,但是有关CodY在调节碳、氮代谢以及抗胁迫中的作用尚未见报道。
通过比较基因组分析发现,嗜热链球菌基因组上存在一段保守的蛋白编码序列codY,推测其编码蛋白CodY具有重要的调控功能。本论文聚焦S. thermophlisST2017全局转录调控因子CodY的碳、氮代谢调控及抗胁迫机制,通过转录组分析,建立CodY对碳、氮代谢的调控网络,利用凝胶阻滞电泳(Electrophoretic Mobility Shift Assay, EMSA)和等温滴定量热法((Isothermal Titration Calorimetry,ITC)分析其调控机制,阐明CodY在抗胁迫中的调控作用,开展工作及取得结果如下：
1、S. thermophlis ST2017全局转录调控因子CodY的调控网络分析
通过比较基因组分析,确定S. thermophlisST2017基因组上的codY基因序列。利用温度敏感型质粒pG+host9,通过同源双交换敲除S. thermophlis ST2017基因组上的codY基因,得到突变株S. thermophlis ST2017△codY。通过转录组测序,分析比较野生株ST2017和突变株ST2017△codY基因差异表达谱,并通过荧光定量PCR验证。结果显示,codY基因敲除后共有87个基因发生了差异表达,其中62个基因的转录受CodY抑制,对这些差异表达基因进行分类,发现CodY调控以下代谢类型的基因转录：(1)控制氨基酸合成与转运：如分支氨基酸的合成(ilvA、LeuABCD和ilvBHC)及转运(livJHMGF),色氨酸(TrpEGDCFBA)、半胱氨酸的合成(cysM2metB2cysE2),天冬氨酸、丝氨酸和苏氨酸的合成,组氨酸分解代谢中的鸟苷水合酶基因(hutU)则是氨基酸代谢中唯一受CodY转录激活的基因；(2)碳代谢相关基因的转录,如EMP途径中的甘油醛3一磷酸脱氢酶和参与半乳糖代谢的磷酸变位酶；(3)CodY对谷氨酸脱氢酶基因gdhA具有强烈的转录抑制作用；(4)控制了多种ABC转运蛋白和二价离子转运蛋白的转录如STND_0202-0203、STND1235-1232和STND_0733;(5)激活次黄嘌呤(IMP)合成操纵子purCLFhNH的转录,并抑制尿嘧啶合成操纵子的转录；(6)参与环境胁迫相关基因的转录调控,如伴侣蛋白DnaK和GroESL操纵子在codY突变株中显著上调,与渗透压胁迫相关的STND_0113、STND0135和与氧胁迫相关的STND_1346则发生了转录下调,同时一些参与环境胁迫应答的转录调控因子也受CodY调控；(7)抑制二组分系统TCS01的转录；(8)其他基因的转录,在差异表达谱中,有大量基因的功能没有确切的注释。由此看出,CodY参与了细胞的多种代谢途径的调控,尤其对氮代谢的调节更为广泛,同时还参与调控环境胁迫相关基因的转录。
2、S. thermophilus ST2017全局转录调控因子CodY的作用机制研究
在革兰氏阳性菌中,转录调控因子CodY通过和细胞内GTP和BCAA的协同作用来调控目的基因的转录,为了确定S. thermophilus ST2017中CodY的作用机制,选取在转录组中明显上调的分支氨基酸转运蛋白操纵子(livJHMG)为研究对象,通过EMSA检测CodY和livJHMG启动子的相互作用。结果显示,CodY和livJHMG启动子具有明显的结合作用,当反应体系中添加2mM的GTP时,CodY和启动子结合能力没有变化,而15mM的BCAA能够有效促进CodY和启动子的结合,说明在S. thermophilus ST2017中,GTP不能促进CodY和启动子的结合,BCAA是唯一的激活效应物,其作用模式类似于乳酸乳球菌(Lactococcus lactis)和肺炎链球菌(Streptococcus pneumoniae)。为了证明效应物BCAA的正调控作用,将livJHMG启动子和绿色荧光蛋白基因融合后在S.thermophilus ST2017中表达。结果显示,BCAA能够有效降低菌体的荧光强度,从体内进一步证明了BCAA作为CodY效应物的正调控作用。
CodY与调控基因的结合具有高度选择性,为了检测CodY与调控基因的结合位点,选取转录组测序中具有显著差异表达的24个基因,通过MEME预测其启动子区域的序列特征,结果发现存在一段保守的DNA序列A[AT]T[AT]TTC[TC]GA[ACT]A[AT]TT,该序列和L. lactis及枯草芽孢杆菌(Bacillus subtilis)的CodY结合域(AATTTTCNGAAAATT)具有高度的序列相似性,推测S. thermophilus ST2017的CodY通过该保守序列和调控基因的启动子结合。为了验证这一假设,通过ITC实验检测CodY和该保守序列DNA的结合能力,结果证明嗜热链球菌CodY和保守序列具有显著的结合作用,并且以二聚体形式和保守序列按照化学计量数1：1结合；将保守序列的碱基突变后,这种结合作用随之消失,说明该保守序列在CodY的识别中必不可少,而BCAA能够有效提高CodY和保守序列的亲和力,使得解离常数Kd从780nM降低到8.33nM。
3、S. thermophilus ST2017转录调控因子CodY在碳、氮代谢转化中的作用
不同于其他乳酸菌,嗜热链球菌具有完整的从丙酮酸到α-酮戊二酸的代谢途径,并且α-酮戊二酸在谷氨酸脱氢酶的作用下可以生成谷氨酸,实现碳代谢和氮代谢的相互转化。转录组学分析发现,S. thermophilus ST2017△codY中谷氨酸脱氢酶基因(gdhA)的转录水平是野生菌株ST2017的3倍,说明转录调控因子CodY对gdhA具有明显的抑制作用。EMSA进一步证明CodY对gdhA启动子具有直接的转录调控作用。体外测定S. thermophilus ST2017及ST2017△codY的谷氨酸脱氢酶酶活,野生菌株的酶活为190U,而突变株为330U,说明codY基因的缺失解除了CodY对gdhA的转录抑制；将codY基因回补到突变株ST2017△codY,并使之高效表达,谷氨酸脱氢酶的酶活降低到100U,进一步证明了CodY对gdhA表达的抑制作用。
在嗜热链球菌中,谷氨酸脱氢酶催化α-酮戊二酸合成谷氨酸是一个可逆反应,但催化两种反应的Km值不同,α-酮戊二酸合成谷氨酸的Km值为0.131±0.030mM,远低于谷氨酸降解为α-酮戊二酸的Km值1.170±0.088mM,这使得在嗜热链球菌体内,具有从α-酮戊二酸合成谷氨酸的代谢优势。控制化学合成培养基(Chemical-defined Medium, CDM)中的氨基酸组分,只添加维持S.thermophilus ST2017生长必须的组氨酸、半胱氨酸、脯氨酸和甲硫氨酸。当乳糖浓度从0.5%提高到1.0%时,菌株ST2017的生物量从0.768±0.011上升到1.048±0.061,而突变株ST2017△codY的生物量则从0.822±0.017增长到1.381±0.023,说明在氮源限制性条件下,菌株生物量的增加来自于乳糖向氨基酸的转化。为了验证谷氨酸脱氢酶在乳糖向氨基酸转化中的作用,分别敲除野生菌株ST2017和突变株ST2017△codY的gdhA基因,得到gdhA突变株ST2017△gdhA和ST2017△codY△gdhA.测定其在0.5%乳糖的CDM中的生物量,分别为0.305±0.034和0.531±0.041,相对菌株ST2017和ST2017△codY显著下降；而乳糖浓度增加到1.0%,生物量只略微提高到0.328±0.009和0.581±0.104。表明在氮源限制条件下,嗜热链球菌通过谷氨酸脱氢酶将充足的碳源转化为氨基酸供菌体生长,codY基因的缺失提高了这种转化能力,当切断碳、氮代谢偶联节点时(缺失gdhA基因),菌体生长受到严重抑制。
在碳源限制条件下,将胰蛋白胨从0.25%提高到0.5%时,菌株ST2017和ST2017△codY的生物量分别提高到1.340±0.067和1.403±0.053,而谷氨酸脱氢酶基因突变株ST2017△gdhA和ST2017△codY△gdhA的生物量只能提高到1.188±0.004和1.164±0.044。说明谷氨酸脱氢酶的失活,使得氮源转化为碳源的能力也降低,最终限制了菌体生长。综合以上结果,可以看出嗜热链球菌全局转录调控因子CodY通过控制谷氨酸脱氢酶来影响碳、氮代谢的相互转化,以此调控菌体的生长。
4、S.thermophilus ST2017转录调控因子CodY在菌体抗胁迫中的作用
嗜热链球菌是重要的乳制品发酵菌株,在发酵剂生产、存放及乳品发酵过程中常常遭受氧、温度、噬菌体等多种胁迫压力,从而导致菌株生长缓慢、发酵能力减弱,直接影响到产品的结构和风味。转录组学分析发现,二组分系统TCS0l在突变株ST2017△codY中显著上调,而TCS01参与压力响应蛋白的转录调控。为了检测因TCS01上调而引起的相关压力响应蛋白的转录变化,构建了二组分系统TCS01过表达菌株ST2017-TCS01exp,比较分析野生菌株ST2017、突变株ST2017△cody及ST2017-TCSOlexp中压力响应蛋白的差异表达。结果显示,嗜热链球菌CodY不但自身可以调控压力响应蛋白DnaK、GroESL STND0113和STND_0135的转录,而且通过控制二组分系统TCS01间接调控STND_1346的转录。
嗜热链球菌是兼性厌氧菌株,可以通过超氧化物歧化酶和NADH氧化酶降低胞内超氧自由基和分子氧浓度,同时利用谷胱甘肽/谷氧还原蛋白维持胞内氧化还原平衡。而谷胱甘肽合成酶基因(STND_1346)的转录水平直接受二组分系统TCS01的抑制,CodY通过控制TCS01来间接调控STND_1346的转录水平。比较S.thermophilus ST2017和ST2017△codY的抗氧胁迫能力。在CDM培养基中添加谷氨酸和甘氨酸,经过H202处理后,ST2017的存活数是突变株的3倍以上；菌株ST2017细胞内谷胱甘肽浓度达到3.40±0.28μ/1,而突变株ST2017△codY为2.47±0.04gM/1；当不添加谷氨酸和甘氨酸时,菌株ST2017和ST2017△codY的存活数都显著下降,没有明显差异,并且胞内基本测不到谷胱甘肽含量。由此说明突变株ST2017△codY抗氧胁迫能力的下降是因为体内谷胱甘肽含量的减少。进一步研究发现,突变株ST2017△codY中,DnaK和GroESL操纵子的转录上调提高了菌体的高温胁迫耐受性；而STND_0113、STND_0135基因的转录下调则降低了菌体对渗透压胁迫的抗性。由此看出,全局转录调控因子CodY在嗜热链球菌对抗氧、温度、渗透压等胁迫压力中具有重要的调控功能。
噬菌体污染是乳制品发酵过程中遇到的不可避免的安全隐患,为此发酵剂菌株自身编码多种防御机制抵抗噬菌体的感染,其中,CRISPR/Cas是最近发现的一种新的对抗外源DNA入侵的免疫机制。转录学分析发现,S. thermophilusST2017转录调控因子CodY对CRISPR1/Cas系统的cas5基因具有强烈的转录抑制作用。研究显示,二组分系统TCS01也抑制cas5的转录。比较S. thermophilusST2017和ST2017△codY中pSEC质粒的丢失率,结果发现无选择压力下传代100代,ST2017△codY的丢失率为8/768,远高于ST2017的1/788。将Cas5基因过表达,外源质粒在S. thermophilusST2017中的丢失率大大升高；同时提高了菌体S. thermophilusST1368对噬菌体的抗性。测序发现,在得到的质粒丢失菌株和噬菌体抗性菌株中,CRISPR序列中并没有新的间隔序列(Spacer)插入,这与已报道的二型CRISPR/Cas系统的作用机制不同。体外测定发现Cas5蛋白具有不依赖于crRNA和tracrRNA的核酸酶活性,能够随意切割λ-DNA和质粒。推测正是Cas5蛋白的核酸酶活性,使得外源质粒在嗜热链球菌CodY缺失突变株和Cas5过表达菌株中的丢失率提高,同时更容易产生对噬菌体的抗性,这是首次发现嗜热链球菌CRISPR/Cas系统中Cas5的非特异性切割引起的对外源DNA的免疫性。
5、基于丙氨酸消旋酶的食品级表达系统的构建及应用
随着遗传操作工具的完善,乳酸菌作为活疫苗载体受到重视。传统表达系统因带有抗生素抗性选择标记而限制其在实际生产中的应用。通过食品级表达体系表达抗原蛋白,使得乳酸菌成为新一代的活菌疫苗。本文以丙氨酸消旋酶基因(alr)为选择标记,分别构建了诱导型和组成型食品级分泌表达体系。首先通过温度敏感型质粒pG+host9和自杀性整合载体pRV300分别敲除了Lactococcus lactisNZ9000和Lactobacillus casei BL23的丙氨酸消旋酶基因,使宿主细胞自身成为D-Ala营养缺陷型,然后以丙氨酸消旋酶基因作为互补选择标记,实现功能回补。以绿色荧光蛋白作为报告基因,与诱导型nisA启动子融合表达,在乳酸乳球菌中构建了严谨的食品级诱导分泌表达体系,实现了猪圆环病毒衣壳蛋白高效的分泌表达,其最大分泌量达到1,059±99μg/1；且nisin诱导2小时,重组菌就能持续分泌衣壳蛋白,到10小时其分泌量依然达到940±101μg/1。同时还将卷曲乳杆菌(Lactobacillus crispatus)S-层蛋白的启动子和核酸酶基因(nuc B)融合,构建了适用于乳酸乳球菌和干酪乳杆菌的组成型食品级分泌表达载体,实现了肠毒性大肠杆菌(Enterotoxigenic Escherichia coli, ETEC)鞭毛蛋白(FaeG)在乳酸乳球菌和干酪乳杆菌中分泌表达。上述构建的食品级表达体系,为乳酸菌活疫苗的开发奠定了基础。
Streptococcus thermophilus is a major dairy starter traditionally used in combination with Lactobacillus delbrueckii subsp. bulgaricus or Lb. helveticus for the manufacture of yogurt and cheeses. S. thermophilus is a "generally recognised as safe"(GRAS) species and over1021live cells are ingested annually. Notably, S. thermophilus forms a unique carbon metabolic system to efficiently utilize the lactose in milk to provide energy for tis growth. S. thermophilus is a fastidious microorganism and requires an exogenous source of amino acids or peptides for optimal growth. As milk is poor in these low-molecular-weight compounds, its growth largely depends on proteolytic system to achieve hydrolysis of caseins. The weak activity or missing of cell envelope proteinase strongly limits the growth. S. thermophilus acquries a well-developed nitrogen pathway for de novo biosynthesis of amino acids by horizontal gene transfer (HGT) due to its adaptation to milk. During dairy processes, S. thermophilus always exposes to reactive O2species (ROS), heat shock, phage infection stresses, and their potential deleterious effects on growth, fermentative capabilities and viability, consequently have repercussions on texture and flavour of the final products. Fortunately, all bacteria have additional layers of control that coordinate the use of nutrients and the resistance to environmental stress using global regulators. CodY is a highly conserved protein in the low-G-C group Gram-positive bacteria, defines a unique family of regulatory proteins. CodY is helpful for bacteria to adaption to poor nutritional availability, and also controls the catabolic pathways, environment response as well as the avirulence of strain. The effects of CodY is stimulated by interaction with either of two ligands, GTP or BCAA. However, the mechanism of CodY regulation possesses strain specificity.
Analysis of the annotated S. thermophilus genome sequences identified a conserved gene designated codY. Here, we focused on the global regulator CodY, and studied the metabolic regulation and stress response of S. thermophilus ST2017. The carbon and nitrogen metabolism regulatory network was elucidated by transcriptome analysis, the regulatory mechanism of CodY was investigated by electrophoretic mobility shift assay (EMSA) and isothermal titration calorimetry (ITC), and clarified the role of CodY in stress response. The detailed contents and results of this thesis were as follows:
The CodY regulon of S. thermophilus ST2017
To identify genes regulated by CodY, the codY gene of S. thermophilus was knockout using the thermosensitive plasmid pG+host9, and generating the mutant ST2017△codY. To establish the extent of the CodY regulon in S. thermophilus ST2017, RNA-seq was undertaken. The transcriptome analysis released87differently expressed genes in ST2017△codY, and62out of87genes were repressed by CodY. These genes were divided into different groups based on the function of regulated genes as follows:(1) Amino acids biosynthesis and transport. The genes involved in biosynthesis of BCAA, tryptophan, cysteine, aspartic acid, serine and threonine synthesis were repressed. The guanosine hydratase gene (hutU), during the histidine degradation process is the sole gene actived by CodY in amino acids metabolism.(2) The global regultor CodY aslo controlled the glyceraldehyde3-phosphate dehydrogenase which is involved in EMP pathway and phosphate mutase which is involved in Leloir pathway.(3)The gdhA gene was significantly derepressed in codY mutant.(4) A number of genes, such as STND_0202-0203、STND_1235-1232and STND_0733involved in ABC transporter and ion channel were derepressed.(5) Transcriptome data showed that CodY also involved in regulation of genes respond to environmental stresses. Chaperone DnaK and GroESL operons in codY mutant were significantly upregulated. Osmotic stress-related STND_0113, STND_0135, and oxidative stress-related STND_1346were downregulated.(6)Meanwhile the two-component system TCS01involved in environmental stress response is also regulated by CodY.(7) CodY actived the transcription of hypoxanthine guanine (IMP) de novo synthesis operon, while the transcription of uracil synthesis operon was repressed.(8) A number of hypothetical proteins were regulated by CodY. In conclusion, the global regulator CodY were involved in the regulation of varieties of metabolic pathways, especially in the fine-turning of nitrogen metabolism. Meanwhile, CodY also regulated the genes responding to environmental stress.
The regulation mechanism of CodY in S. thermophilus ST2017
In Gram-positive bacteria, the regulation CodY binds the DNA fragment with the help of the affecters GTP and BCAA. To find out the affecter CodY needed in S. thermophilus ST2017, the promoter of livJHMG operon identified by transcriptome analysis was PCR amplified, and the PCR product was mixed with purified CodY to perform out the EMSA analysis. This binding ability of CodY was enhanced by addition of15mM BCAA, do not of GTP. The EMSA expriments showed that the S. thermophilus CodY responded to the intracellular BCAA concentrations but not to physiological fluctuations in intracellular GTP. To further confirm the positive effect of BCAA, the livJHMG promoter Pliv was fused with green fluorescent protein gene (gfp) and the plasmid pSEC-Pliv-gfp was expressed in S. thermophilus ST2017. As a result, the level of fluorescence intensity was reduced by addition of BCAA, and this confirmed the positive effect of BCAA in vivo. A15-bp conserved motif A[AT]T [AT]TTC[TC] GA[ACT][AT]TT in the promoter regions of CodY regulated genes was identified using MEME soft. Furthermore, there was a high similarity to the canonical CodY-box, AATTTTCWGAAAATT, which was previously described based on analysis of L. lactis and B. subtilis CodY-regulated genes. The interaction of CodY and DNA containing the15-bp conserved motif was monitored by ITC analysis. The results proved that CodY and conserved motif had a significant combination effect, indicating that the conserved sequence has a crucial role in the identification of CodY. BCAA could effectively improve the affinity, and the dissociation constant Kd reduced from780nM to8.33nM.
The global regulator CodY coordinates the flow of carbon and nitrogen metabolism
S. thermophilus has a complete metabolic pathway from pyruvate to a-oxoglutarate, and the metabolite a-oxoglutarate stands at the crossroads between carbon metabolism and nitrogen metabolism. As one of the substrates of glutamate dehydrogenase (gdhA), a-oxoglutarate provides the de novo carbon skeleton for glutamate. Moreover, as the product of glutamate metabolism by glutamate dehydrogenase, a-oxoglutarate can be the entry point into central metabolism for the carbon skeletons of several amino acids. The transcriptomics and real-time PCR showed that glutamate dehydrogenase gene (gdhA) was significantly repressed by CodY. EMSA further confirmed that CodY had a direct role in the regulation of gdhA transcription. The glutamate dehydrogenase enzyme activity of S. thermophilus ST2017was190U, and deletion of codY increased the enzyme activity to330U, while overexpression of codY in S. thermophilus ST2017△codY decreased the enzyme activity to110U. The results showed that CodY directly inhibited the expression of gdhA. In S. thermophilus ST2017, the glutamate dehydrogenase catalyzed both a-ketoglutarate synthesis to glutamic acid with a Km value of0.131±0.030mM, and glutamic acid degradation to a-ketoglutarate with a Km value of1.170±0.088mM. The Km value of a-oxoglutarate to glutamate synthesis was much lower than that of glutamate to a-oxoglutarate. This metabolic advange forced the carbon into nitrogen metabolism, and effectively utilized the lactose in milk. Histidine, cysteine, proline and methionine were added to Chemical-defined medium (CDM) to maintain the growth of S. thermophilus ST2017. When the lactose concentration in CDM increased from0.5%to1.0%, the biomass increased from0.768±0.011to1.048±0.061in S. thermophilus ST2017, while the biomass of ST2017△codY rised from0.822±0.017to1.381±0.023. We supposed that the increased biomass was from the conversion of lactose to amino acids, under nitrogen limiting condition. To verify the role of glutamate dehydrogenase in the conversion of lactose to amino acids, the gdhA gene was deleted in S. thermophilus ST2017and ST2017△codY, yielding ST2017△gdhA and ST2017△codY△gdhA, and the biomass decreased to0.305±0.034and0.531±0.041in CDM with0.5%lactose. The biomass only slightly increased to0.328±0.009and0.581±0.104with the increase of lactose to1.0%. The results showed that, under nitrogen limiting condition, S. thermophilus can turn carbon source into amino acids for cell growth through glutamate dehydrogenase, the deletion of codY increased this transformation capability. When the link between carbon and nitrogen metabolism was blocked(inactive gdhA gene), the biomass was severely decreased.
In carbon limiting condition, the biomass of S. thermophilus ST2017and ST2017△codY increased to1.340±0.067and1.403±0.053when the nitrogen (Tryptone) increased from0.25%to0.5%, while the biomass of ST2017△gdhA and ST2017△codY△gdhA was only increased to1.188±0.004and1.164±0.044. This result confirmed that the capacity of converting carbon to nitrogen was also reduced in AgdhA mutant, and eventually limit the cell growth. This chapter elaborated on the interconversion of carbon, nitrogen metabolism in S. thermophilus by global regulator CodY.
The role of CodY in response to environmental stress
The ability of S. thermophilus to cope with the environmental stresses during manufacture and preservation (oxidative stress, high temperature, and phage infection) is essential for its performance as a starter. This research found that S. thermophilus ST2017global regulator CodY responded to environmental stress by controlling the two-component system TCS01, while CodY itself can regulate various stress response proteins. The results showed that CodY directly regulated the transcription of DnaK, GroESL, STND_0113and STND_0135, while the transcription of STND_1346gene was regulated by the two-component system TCS01directly.
S. thermophilus is a facultative anaerobe, and reduces the intracellular concentration of superoxide radicals and oxygen molecules by superoxide dismutase and NADH oxidase. The glutathione (GSH/Gr/Grx) system is responsible for the bacterial intracellular low redox potential and the maintenance of proteins in their reduced state. To enhance bacterial resistance to reactive oxygen, the glutathione synthetase gene (STND_1346) was derepressed in ST2017△coY, while the glutathione synthetase gene(STND_1346) was directly controlled by the two-component system TCS01. The global regulator CodY controlled the expression of STND_1346indirectly by repressing the two-component system TCS01. Comparing oxidative stress tolerance of S. thermophilus ST2017and ST2017△codY After H2O2treatment, the survival of ST2017is more than3-fold than that of ST2017△codY in CDM containing glutamate and glycine, and the concentration of glutathione was3.40±0.28μM in ST2017and2.47±0.04μM in ST2017△codY. The survival of ST2017and ST2017△codY was significantly decreased in CDM without glutamate and glycine, and the concentration of glutathione was basically undetectable. The results showed that, the susceptible to oxidative stress of ST2017△codY compared to ST2017was due to the decrease of glutathione concentration. Further research showed that, the upregulation of DnaK and GroESL in codY mutant significantly improved the heat shock tolerance, while the downregulation of STND_0113and STND_0135genes reduced the osmotic stress resistance. These results showed that the global regulator CodY was responded to oxidative, temperature, osmotic stress, and had an important regulatory function in S. thermophilus ST2017.
The systematic use of the same S. thermophilus strains in dairy processes has been impaired by the ubiquitous presence of virulent phages. This research found that the cas5gene in S. thermophilus CRISPR1/Cas system was strongly repressed by the global regulator CodY and two-component system TCS01directly. The over-expression of cas5gene greatly increased the loss rate of exogenous plasmid in S. thermophilus ST2017, while increasing bacterial resistance to phage. Further studies showed that no new spacer insertion was found in CRISPR sequences, this was different from the mechanism of type Ⅱ CRISPR/Cas systems. Furthermore, we found that Cas5protein had a tracrRNA and crRNA independent nuclease activity in vitro assay, and could cut the plasmid and λ-DNA with a non-specific manner. We speculated that the nuclease activity of Cas5protein increased the loss rate of exogenous plasmid, and mad it easier to generate phage immunity. This was the first evidence of S. thermophilus CRISPR/Cas system to immunize exogenous DNA by non-specific nuclease activity of Cas5.
Construction and application of food-grade expression system for lactis acid bacteria based on alanine racemase gene
Lactic acid bacteria (LAB) are widely used as probiotics and have great potential to serve as antigen delivery vehicle for oral vaccines. However, the application of genetically modified organisms in food products and practical vaccines requires safe and sustainable genetic tools devoid of any antibiotic-resistance markers. Therefore, sophisticated food-grade marker systems are being developed. Here, we focused on the air gene as a selection marker, and an inducible food-grade system as well as a constitutive food-grade system were constructed with DNA fragments entirely from lactic acid bacteria. The new inducible food-grade expression system in L. lactis with pSEC replicon, PnisA promoter and air selection marker was constructed. The green fluorescence protein (gfp) was used as a reporter for gene expression to verify the feasibility of the new host/vector system. We, therefore, focused on the lactococcal strain as a cell factory, which was used to express high amount of the capsid protein of porcine circovirus type Ⅱ(dCap). The maximum amount of dCap reached1,059±99μg/1, and after a2h nisin pulse induction, the dCap protein maintained secretion even10h after the nisin pulse, and decreased to940±101μg l-1. The results verified the potential application of the new inducible food-grade system for an oral vaccine purpose. Enterotoxigenic Escherichia coli (ETEC) strains are the major cause of diarrhea in neonatal piglets. The fimbriae as colonizing factors in the pathogenesis of ETEC constitute a primary target for vaccination against ETEC. Considering of the safety of the genetic modified LAB, a constitutive food-grade secretion system was constructed with promoter of S-layer protein, replicon and selection marker alanine racemase(alr). To evaluate the feasibility of the system, Nuclease (NucB) from Staphylococcus aureus was used as a reporter gene to express in both L. lactis and Lb. casei. Subsequently, extracellular expression of fimbrial adhesin FaeG of ETEC was confirmed by western blot analysis. These two new systems were real food-grade system as appropriate antigen delivery systems, and also suitable for the production of ingredients in food industry.
通过比较基因组分析发现,嗜热链球菌基因组上存在一段保守的蛋白编码序列codY,推测其编码蛋白CodY具有重要的调控功能。本论文聚焦S. thermophlisST2017全局转录调控因子CodY的碳、氮代谢调控及抗胁迫机制,通过转录组分析,建立CodY对碳、氮代谢的调控网络,利用凝胶阻滞电泳(Electrophoretic Mobility Shift Assay, EMSA)和等温滴定量热法((Isothermal Titration Calorimetry,ITC)分析其调控机制,阐明CodY在抗胁迫中的调控作用,开展工作及取得结果如下：
1、S. thermophlis ST2017全局转录调控因子CodY的调控网络分析
通过比较基因组分析,确定S. thermophlisST2017基因组上的codY基因序列。利用温度敏感型质粒pG+host9,通过同源双交换敲除S. thermophlis ST2017基因组上的codY基因,得到突变株S. thermophlis ST2017△codY。通过转录组测序,分析比较野生株ST2017和突变株ST2017△codY基因差异表达谱,并通过荧光定量PCR验证。结果显示,codY基因敲除后共有87个基因发生了差异表达,其中62个基因的转录受CodY抑制,对这些差异表达基因进行分类,发现CodY调控以下代谢类型的基因转录：(1)控制氨基酸合成与转运：如分支氨基酸的合成(ilvA、LeuABCD和ilvBHC)及转运(livJHMGF),色氨酸(TrpEGDCFBA)、半胱氨酸的合成(cysM2metB2cysE2),天冬氨酸、丝氨酸和苏氨酸的合成,组氨酸分解代谢中的鸟苷水合酶基因(hutU)则是氨基酸代谢中唯一受CodY转录激活的基因；(2)碳代谢相关基因的转录,如EMP途径中的甘油醛3一磷酸脱氢酶和参与半乳糖代谢的磷酸变位酶；(3)CodY对谷氨酸脱氢酶基因gdhA具有强烈的转录抑制作用；(4)控制了多种ABC转运蛋白和二价离子转运蛋白的转录如STND_0202-0203、STND1235-1232和STND_0733;(5)激活次黄嘌呤(IMP)合成操纵子purCLFhNH的转录,并抑制尿嘧啶合成操纵子的转录；(6)参与环境胁迫相关基因的转录调控,如伴侣蛋白DnaK和GroESL操纵子在codY突变株中显著上调,与渗透压胁迫相关的STND_0113、STND0135和与氧胁迫相关的STND_1346则发生了转录下调,同时一些参与环境胁迫应答的转录调控因子也受CodY调控；(7)抑制二组分系统TCS01的转录；(8)其他基因的转录,在差异表达谱中,有大量基因的功能没有确切的注释。由此看出,CodY参与了细胞的多种代谢途径的调控,尤其对氮代谢的调节更为广泛,同时还参与调控环境胁迫相关基因的转录。
2、S. thermophilus ST2017全局转录调控因子CodY的作用机制研究
在革兰氏阳性菌中,转录调控因子CodY通过和细胞内GTP和BCAA的协同作用来调控目的基因的转录,为了确定S. thermophilus ST2017中CodY的作用机制,选取在转录组中明显上调的分支氨基酸转运蛋白操纵子(livJHMG)为研究对象,通过EMSA检测CodY和livJHMG启动子的相互作用。结果显示,CodY和livJHMG启动子具有明显的结合作用,当反应体系中添加2mM的GTP时,CodY和启动子结合能力没有变化,而15mM的BCAA能够有效促进CodY和启动子的结合,说明在S. thermophilus ST2017中,GTP不能促进CodY和启动子的结合,BCAA是唯一的激活效应物,其作用模式类似于乳酸乳球菌(Lactococcus lactis)和肺炎链球菌(Streptococcus pneumoniae)。为了证明效应物BCAA的正调控作用,将livJHMG启动子和绿色荧光蛋白基因融合后在S.thermophilus ST2017中表达。结果显示,BCAA能够有效降低菌体的荧光强度,从体内进一步证明了BCAA作为CodY效应物的正调控作用。
CodY与调控基因的结合具有高度选择性,为了检测CodY与调控基因的结合位点,选取转录组测序中具有显著差异表达的24个基因,通过MEME预测其启动子区域的序列特征,结果发现存在一段保守的DNA序列A[AT]T[AT]TTC[TC]GA[ACT]A[AT]TT,该序列和L. lactis及枯草芽孢杆菌(Bacillus subtilis)的CodY结合域(AATTTTCNGAAAATT)具有高度的序列相似性,推测S. thermophilus ST2017的CodY通过该保守序列和调控基因的启动子结合。为了验证这一假设,通过ITC实验检测CodY和该保守序列DNA的结合能力,结果证明嗜热链球菌CodY和保守序列具有显著的结合作用,并且以二聚体形式和保守序列按照化学计量数1：1结合；将保守序列的碱基突变后,这种结合作用随之消失,说明该保守序列在CodY的识别中必不可少,而BCAA能够有效提高CodY和保守序列的亲和力,使得解离常数Kd从780nM降低到8.33nM。
3、S. thermophilus ST2017转录调控因子CodY在碳、氮代谢转化中的作用
不同于其他乳酸菌,嗜热链球菌具有完整的从丙酮酸到α-酮戊二酸的代谢途径,并且α-酮戊二酸在谷氨酸脱氢酶的作用下可以生成谷氨酸,实现碳代谢和氮代谢的相互转化。转录组学分析发现,S. thermophilus ST2017△codY中谷氨酸脱氢酶基因(gdhA)的转录水平是野生菌株ST2017的3倍,说明转录调控因子CodY对gdhA具有明显的抑制作用。EMSA进一步证明CodY对gdhA启动子具有直接的转录调控作用。体外测定S. thermophilus ST2017及ST2017△codY的谷氨酸脱氢酶酶活,野生菌株的酶活为190U,而突变株为330U,说明codY基因的缺失解除了CodY对gdhA的转录抑制；将codY基因回补到突变株ST2017△codY,并使之高效表达,谷氨酸脱氢酶的酶活降低到100U,进一步证明了CodY对gdhA表达的抑制作用。
在嗜热链球菌中,谷氨酸脱氢酶催化α-酮戊二酸合成谷氨酸是一个可逆反应,但催化两种反应的Km值不同,α-酮戊二酸合成谷氨酸的Km值为0.131±0.030mM,远低于谷氨酸降解为α-酮戊二酸的Km值1.170±0.088mM,这使得在嗜热链球菌体内,具有从α-酮戊二酸合成谷氨酸的代谢优势。控制化学合成培养基(Chemical-defined Medium, CDM)中的氨基酸组分,只添加维持S.thermophilus ST2017生长必须的组氨酸、半胱氨酸、脯氨酸和甲硫氨酸。当乳糖浓度从0.5%提高到1.0%时,菌株ST2017的生物量从0.768±0.011上升到1.048±0.061,而突变株ST2017△codY的生物量则从0.822±0.017增长到1.381±0.023,说明在氮源限制性条件下,菌株生物量的增加来自于乳糖向氨基酸的转化。为了验证谷氨酸脱氢酶在乳糖向氨基酸转化中的作用,分别敲除野生菌株ST2017和突变株ST2017△codY的gdhA基因,得到gdhA突变株ST2017△gdhA和ST2017△codY△gdhA.测定其在0.5%乳糖的CDM中的生物量,分别为0.305±0.034和0.531±0.041,相对菌株ST2017和ST2017△codY显著下降；而乳糖浓度增加到1.0%,生物量只略微提高到0.328±0.009和0.581±0.104。表明在氮源限制条件下,嗜热链球菌通过谷氨酸脱氢酶将充足的碳源转化为氨基酸供菌体生长,codY基因的缺失提高了这种转化能力,当切断碳、氮代谢偶联节点时(缺失gdhA基因),菌体生长受到严重抑制。
在碳源限制条件下,将胰蛋白胨从0.25%提高到0.5%时,菌株ST2017和ST2017△codY的生物量分别提高到1.340±0.067和1.403±0.053,而谷氨酸脱氢酶基因突变株ST2017△gdhA和ST2017△codY△gdhA的生物量只能提高到1.188±0.004和1.164±0.044。说明谷氨酸脱氢酶的失活,使得氮源转化为碳源的能力也降低,最终限制了菌体生长。综合以上结果,可以看出嗜热链球菌全局转录调控因子CodY通过控制谷氨酸脱氢酶来影响碳、氮代谢的相互转化,以此调控菌体的生长。
4、S.thermophilus ST2017转录调控因子CodY在菌体抗胁迫中的作用
嗜热链球菌是重要的乳制品发酵菌株,在发酵剂生产、存放及乳品发酵过程中常常遭受氧、温度、噬菌体等多种胁迫压力,从而导致菌株生长缓慢、发酵能力减弱,直接影响到产品的结构和风味。转录组学分析发现,二组分系统TCS0l在突变株ST2017△codY中显著上调,而TCS01参与压力响应蛋白的转录调控。为了检测因TCS01上调而引起的相关压力响应蛋白的转录变化,构建了二组分系统TCS01过表达菌株ST2017-TCS01exp,比较分析野生菌株ST2017、突变株ST2017△cody及ST2017-TCSOlexp中压力响应蛋白的差异表达。结果显示,嗜热链球菌CodY不但自身可以调控压力响应蛋白DnaK、GroESL STND0113和STND_0135的转录,而且通过控制二组分系统TCS01间接调控STND_1346的转录。
嗜热链球菌是兼性厌氧菌株,可以通过超氧化物歧化酶和NADH氧化酶降低胞内超氧自由基和分子氧浓度,同时利用谷胱甘肽/谷氧还原蛋白维持胞内氧化还原平衡。而谷胱甘肽合成酶基因(STND_1346)的转录水平直接受二组分系统TCS01的抑制,CodY通过控制TCS01来间接调控STND_1346的转录水平。比较S.thermophilus ST2017和ST2017△codY的抗氧胁迫能力。在CDM培养基中添加谷氨酸和甘氨酸,经过H202处理后,ST2017的存活数是突变株的3倍以上；菌株ST2017细胞内谷胱甘肽浓度达到3.40±0.28μ/1,而突变株ST2017△codY为2.47±0.04gM/1；当不添加谷氨酸和甘氨酸时,菌株ST2017和ST2017△codY的存活数都显著下降,没有明显差异,并且胞内基本测不到谷胱甘肽含量。由此说明突变株ST2017△codY抗氧胁迫能力的下降是因为体内谷胱甘肽含量的减少。进一步研究发现,突变株ST2017△codY中,DnaK和GroESL操纵子的转录上调提高了菌体的高温胁迫耐受性；而STND_0113、STND_0135基因的转录下调则降低了菌体对渗透压胁迫的抗性。由此看出,全局转录调控因子CodY在嗜热链球菌对抗氧、温度、渗透压等胁迫压力中具有重要的调控功能。
噬菌体污染是乳制品发酵过程中遇到的不可避免的安全隐患,为此发酵剂菌株自身编码多种防御机制抵抗噬菌体的感染,其中,CRISPR/Cas是最近发现的一种新的对抗外源DNA入侵的免疫机制。转录学分析发现,S. thermophilusST2017转录调控因子CodY对CRISPR1/Cas系统的cas5基因具有强烈的转录抑制作用。研究显示,二组分系统TCS01也抑制cas5的转录。比较S. thermophilusST2017和ST2017△codY中pSEC质粒的丢失率,结果发现无选择压力下传代100代,ST2017△codY的丢失率为8/768,远高于ST2017的1/788。将Cas5基因过表达,外源质粒在S. thermophilusST2017中的丢失率大大升高；同时提高了菌体S. thermophilusST1368对噬菌体的抗性。测序发现,在得到的质粒丢失菌株和噬菌体抗性菌株中,CRISPR序列中并没有新的间隔序列(Spacer)插入,这与已报道的二型CRISPR/Cas系统的作用机制不同。体外测定发现Cas5蛋白具有不依赖于crRNA和tracrRNA的核酸酶活性,能够随意切割λ-DNA和质粒。推测正是Cas5蛋白的核酸酶活性,使得外源质粒在嗜热链球菌CodY缺失突变株和Cas5过表达菌株中的丢失率提高,同时更容易产生对噬菌体的抗性,这是首次发现嗜热链球菌CRISPR/Cas系统中Cas5的非特异性切割引起的对外源DNA的免疫性。
5、基于丙氨酸消旋酶的食品级表达系统的构建及应用
随着遗传操作工具的完善,乳酸菌作为活疫苗载体受到重视。传统表达系统因带有抗生素抗性选择标记而限制其在实际生产中的应用。通过食品级表达体系表达抗原蛋白,使得乳酸菌成为新一代的活菌疫苗。本文以丙氨酸消旋酶基因(alr)为选择标记,分别构建了诱导型和组成型食品级分泌表达体系。首先通过温度敏感型质粒pG+host9和自杀性整合载体pRV300分别敲除了Lactococcus lactisNZ9000和Lactobacillus casei BL23的丙氨酸消旋酶基因,使宿主细胞自身成为D-Ala营养缺陷型,然后以丙氨酸消旋酶基因作为互补选择标记,实现功能回补。以绿色荧光蛋白作为报告基因,与诱导型nisA启动子融合表达,在乳酸乳球菌中构建了严谨的食品级诱导分泌表达体系,实现了猪圆环病毒衣壳蛋白高效的分泌表达,其最大分泌量达到1,059±99μg/1；且nisin诱导2小时,重组菌就能持续分泌衣壳蛋白,到10小时其分泌量依然达到940±101μg/1。同时还将卷曲乳杆菌(Lactobacillus crispatus)S-层蛋白的启动子和核酸酶基因(nuc B)融合,构建了适用于乳酸乳球菌和干酪乳杆菌的组成型食品级分泌表达载体,实现了肠毒性大肠杆菌(Enterotoxigenic Escherichia coli, ETEC)鞭毛蛋白(FaeG)在乳酸乳球菌和干酪乳杆菌中分泌表达。上述构建的食品级表达体系,为乳酸菌活疫苗的开发奠定了基础。
Streptococcus thermophilus is a major dairy starter traditionally used in combination with Lactobacillus delbrueckii subsp. bulgaricus or Lb. helveticus for the manufacture of yogurt and cheeses. S. thermophilus is a "generally recognised as safe"(GRAS) species and over1021live cells are ingested annually. Notably, S. thermophilus forms a unique carbon metabolic system to efficiently utilize the lactose in milk to provide energy for tis growth. S. thermophilus is a fastidious microorganism and requires an exogenous source of amino acids or peptides for optimal growth. As milk is poor in these low-molecular-weight compounds, its growth largely depends on proteolytic system to achieve hydrolysis of caseins. The weak activity or missing of cell envelope proteinase strongly limits the growth. S. thermophilus acquries a well-developed nitrogen pathway for de novo biosynthesis of amino acids by horizontal gene transfer (HGT) due to its adaptation to milk. During dairy processes, S. thermophilus always exposes to reactive O2species (ROS), heat shock, phage infection stresses, and their potential deleterious effects on growth, fermentative capabilities and viability, consequently have repercussions on texture and flavour of the final products. Fortunately, all bacteria have additional layers of control that coordinate the use of nutrients and the resistance to environmental stress using global regulators. CodY is a highly conserved protein in the low-G-C group Gram-positive bacteria, defines a unique family of regulatory proteins. CodY is helpful for bacteria to adaption to poor nutritional availability, and also controls the catabolic pathways, environment response as well as the avirulence of strain. The effects of CodY is stimulated by interaction with either of two ligands, GTP or BCAA. However, the mechanism of CodY regulation possesses strain specificity.
Analysis of the annotated S. thermophilus genome sequences identified a conserved gene designated codY. Here, we focused on the global regulator CodY, and studied the metabolic regulation and stress response of S. thermophilus ST2017. The carbon and nitrogen metabolism regulatory network was elucidated by transcriptome analysis, the regulatory mechanism of CodY was investigated by electrophoretic mobility shift assay (EMSA) and isothermal titration calorimetry (ITC), and clarified the role of CodY in stress response. The detailed contents and results of this thesis were as follows:
The CodY regulon of S. thermophilus ST2017
To identify genes regulated by CodY, the codY gene of S. thermophilus was knockout using the thermosensitive plasmid pG+host9, and generating the mutant ST2017△codY. To establish the extent of the CodY regulon in S. thermophilus ST2017, RNA-seq was undertaken. The transcriptome analysis released87differently expressed genes in ST2017△codY, and62out of87genes were repressed by CodY. These genes were divided into different groups based on the function of regulated genes as follows:(1) Amino acids biosynthesis and transport. The genes involved in biosynthesis of BCAA, tryptophan, cysteine, aspartic acid, serine and threonine synthesis were repressed. The guanosine hydratase gene (hutU), during the histidine degradation process is the sole gene actived by CodY in amino acids metabolism.(2) The global regultor CodY aslo controlled the glyceraldehyde3-phosphate dehydrogenase which is involved in EMP pathway and phosphate mutase which is involved in Leloir pathway.(3)The gdhA gene was significantly derepressed in codY mutant.(4) A number of genes, such as STND_0202-0203、STND_1235-1232and STND_0733involved in ABC transporter and ion channel were derepressed.(5) Transcriptome data showed that CodY also involved in regulation of genes respond to environmental stresses. Chaperone DnaK and GroESL operons in codY mutant were significantly upregulated. Osmotic stress-related STND_0113, STND_0135, and oxidative stress-related STND_1346were downregulated.(6)Meanwhile the two-component system TCS01involved in environmental stress response is also regulated by CodY.(7) CodY actived the transcription of hypoxanthine guanine (IMP) de novo synthesis operon, while the transcription of uracil synthesis operon was repressed.(8) A number of hypothetical proteins were regulated by CodY. In conclusion, the global regulator CodY were involved in the regulation of varieties of metabolic pathways, especially in the fine-turning of nitrogen metabolism. Meanwhile, CodY also regulated the genes responding to environmental stress.
The regulation mechanism of CodY in S. thermophilus ST2017
In Gram-positive bacteria, the regulation CodY binds the DNA fragment with the help of the affecters GTP and BCAA. To find out the affecter CodY needed in S. thermophilus ST2017, the promoter of livJHMG operon identified by transcriptome analysis was PCR amplified, and the PCR product was mixed with purified CodY to perform out the EMSA analysis. This binding ability of CodY was enhanced by addition of15mM BCAA, do not of GTP. The EMSA expriments showed that the S. thermophilus CodY responded to the intracellular BCAA concentrations but not to physiological fluctuations in intracellular GTP. To further confirm the positive effect of BCAA, the livJHMG promoter Pliv was fused with green fluorescent protein gene (gfp) and the plasmid pSEC-Pliv-gfp was expressed in S. thermophilus ST2017. As a result, the level of fluorescence intensity was reduced by addition of BCAA, and this confirmed the positive effect of BCAA in vivo. A15-bp conserved motif A[AT]T [AT]TTC[TC] GA[ACT][AT]TT in the promoter regions of CodY regulated genes was identified using MEME soft. Furthermore, there was a high similarity to the canonical CodY-box, AATTTTCWGAAAATT, which was previously described based on analysis of L. lactis and B. subtilis CodY-regulated genes. The interaction of CodY and DNA containing the15-bp conserved motif was monitored by ITC analysis. The results proved that CodY and conserved motif had a significant combination effect, indicating that the conserved sequence has a crucial role in the identification of CodY. BCAA could effectively improve the affinity, and the dissociation constant Kd reduced from780nM to8.33nM.
The global regulator CodY coordinates the flow of carbon and nitrogen metabolism
S. thermophilus has a complete metabolic pathway from pyruvate to a-oxoglutarate, and the metabolite a-oxoglutarate stands at the crossroads between carbon metabolism and nitrogen metabolism. As one of the substrates of glutamate dehydrogenase (gdhA), a-oxoglutarate provides the de novo carbon skeleton for glutamate. Moreover, as the product of glutamate metabolism by glutamate dehydrogenase, a-oxoglutarate can be the entry point into central metabolism for the carbon skeletons of several amino acids. The transcriptomics and real-time PCR showed that glutamate dehydrogenase gene (gdhA) was significantly repressed by CodY. EMSA further confirmed that CodY had a direct role in the regulation of gdhA transcription. The glutamate dehydrogenase enzyme activity of S. thermophilus ST2017was190U, and deletion of codY increased the enzyme activity to330U, while overexpression of codY in S. thermophilus ST2017△codY decreased the enzyme activity to110U. The results showed that CodY directly inhibited the expression of gdhA. In S. thermophilus ST2017, the glutamate dehydrogenase catalyzed both a-ketoglutarate synthesis to glutamic acid with a Km value of0.131±0.030mM, and glutamic acid degradation to a-ketoglutarate with a Km value of1.170±0.088mM. The Km value of a-oxoglutarate to glutamate synthesis was much lower than that of glutamate to a-oxoglutarate. This metabolic advange forced the carbon into nitrogen metabolism, and effectively utilized the lactose in milk. Histidine, cysteine, proline and methionine were added to Chemical-defined medium (CDM) to maintain the growth of S. thermophilus ST2017. When the lactose concentration in CDM increased from0.5%to1.0%, the biomass increased from0.768±0.011to1.048±0.061in S. thermophilus ST2017, while the biomass of ST2017△codY rised from0.822±0.017to1.381±0.023. We supposed that the increased biomass was from the conversion of lactose to amino acids, under nitrogen limiting condition. To verify the role of glutamate dehydrogenase in the conversion of lactose to amino acids, the gdhA gene was deleted in S. thermophilus ST2017and ST2017△codY, yielding ST2017△gdhA and ST2017△codY△gdhA, and the biomass decreased to0.305±0.034and0.531±0.041in CDM with0.5%lactose. The biomass only slightly increased to0.328±0.009and0.581±0.104with the increase of lactose to1.0%. The results showed that, under nitrogen limiting condition, S. thermophilus can turn carbon source into amino acids for cell growth through glutamate dehydrogenase, the deletion of codY increased this transformation capability. When the link between carbon and nitrogen metabolism was blocked(inactive gdhA gene), the biomass was severely decreased.
In carbon limiting condition, the biomass of S. thermophilus ST2017and ST2017△codY increased to1.340±0.067and1.403±0.053when the nitrogen (Tryptone) increased from0.25%to0.5%, while the biomass of ST2017△gdhA and ST2017△codY△gdhA was only increased to1.188±0.004and1.164±0.044. This result confirmed that the capacity of converting carbon to nitrogen was also reduced in AgdhA mutant, and eventually limit the cell growth. This chapter elaborated on the interconversion of carbon, nitrogen metabolism in S. thermophilus by global regulator CodY.
The role of CodY in response to environmental stress
The ability of S. thermophilus to cope with the environmental stresses during manufacture and preservation (oxidative stress, high temperature, and phage infection) is essential for its performance as a starter. This research found that S. thermophilus ST2017global regulator CodY responded to environmental stress by controlling the two-component system TCS01, while CodY itself can regulate various stress response proteins. The results showed that CodY directly regulated the transcription of DnaK, GroESL, STND_0113and STND_0135, while the transcription of STND_1346gene was regulated by the two-component system TCS01directly.
S. thermophilus is a facultative anaerobe, and reduces the intracellular concentration of superoxide radicals and oxygen molecules by superoxide dismutase and NADH oxidase. The glutathione (GSH/Gr/Grx) system is responsible for the bacterial intracellular low redox potential and the maintenance of proteins in their reduced state. To enhance bacterial resistance to reactive oxygen, the glutathione synthetase gene (STND_1346) was derepressed in ST2017△coY, while the glutathione synthetase gene(STND_1346) was directly controlled by the two-component system TCS01. The global regulator CodY controlled the expression of STND_1346indirectly by repressing the two-component system TCS01. Comparing oxidative stress tolerance of S. thermophilus ST2017and ST2017△codY After H2O2treatment, the survival of ST2017is more than3-fold than that of ST2017△codY in CDM containing glutamate and glycine, and the concentration of glutathione was3.40±0.28μM in ST2017and2.47±0.04μM in ST2017△codY. The survival of ST2017and ST2017△codY was significantly decreased in CDM without glutamate and glycine, and the concentration of glutathione was basically undetectable. The results showed that, the susceptible to oxidative stress of ST2017△codY compared to ST2017was due to the decrease of glutathione concentration. Further research showed that, the upregulation of DnaK and GroESL in codY mutant significantly improved the heat shock tolerance, while the downregulation of STND_0113and STND_0135genes reduced the osmotic stress resistance. These results showed that the global regulator CodY was responded to oxidative, temperature, osmotic stress, and had an important regulatory function in S. thermophilus ST2017.
The systematic use of the same S. thermophilus strains in dairy processes has been impaired by the ubiquitous presence of virulent phages. This research found that the cas5gene in S. thermophilus CRISPR1/Cas system was strongly repressed by the global regulator CodY and two-component system TCS01directly. The over-expression of cas5gene greatly increased the loss rate of exogenous plasmid in S. thermophilus ST2017, while increasing bacterial resistance to phage. Further studies showed that no new spacer insertion was found in CRISPR sequences, this was different from the mechanism of type Ⅱ CRISPR/Cas systems. Furthermore, we found that Cas5protein had a tracrRNA and crRNA independent nuclease activity in vitro assay, and could cut the plasmid and λ-DNA with a non-specific manner. We speculated that the nuclease activity of Cas5protein increased the loss rate of exogenous plasmid, and mad it easier to generate phage immunity. This was the first evidence of S. thermophilus CRISPR/Cas system to immunize exogenous DNA by non-specific nuclease activity of Cas5.
Construction and application of food-grade expression system for lactis acid bacteria based on alanine racemase gene
Lactic acid bacteria (LAB) are widely used as probiotics and have great potential to serve as antigen delivery vehicle for oral vaccines. However, the application of genetically modified organisms in food products and practical vaccines requires safe and sustainable genetic tools devoid of any antibiotic-resistance markers. Therefore, sophisticated food-grade marker systems are being developed. Here, we focused on the air gene as a selection marker, and an inducible food-grade system as well as a constitutive food-grade system were constructed with DNA fragments entirely from lactic acid bacteria. The new inducible food-grade expression system in L. lactis with pSEC replicon, PnisA promoter and air selection marker was constructed. The green fluorescence protein (gfp) was used as a reporter for gene expression to verify the feasibility of the new host/vector system. We, therefore, focused on the lactococcal strain as a cell factory, which was used to express high amount of the capsid protein of porcine circovirus type Ⅱ(dCap). The maximum amount of dCap reached1,059±99μg/1, and after a2h nisin pulse induction, the dCap protein maintained secretion even10h after the nisin pulse, and decreased to940±101μg l-1. The results verified the potential application of the new inducible food-grade system for an oral vaccine purpose. Enterotoxigenic Escherichia coli (ETEC) strains are the major cause of diarrhea in neonatal piglets. The fimbriae as colonizing factors in the pathogenesis of ETEC constitute a primary target for vaccination against ETEC. Considering of the safety of the genetic modified LAB, a constitutive food-grade secretion system was constructed with promoter of S-layer protein, replicon and selection marker alanine racemase(alr). To evaluate the feasibility of the system, Nuclease (NucB) from Staphylococcus aureus was used as a reporter gene to express in both L. lactis and Lb. casei. Subsequently, extracellular expression of fimbrial adhesin FaeG of ETEC was confirmed by western blot analysis. These two new systems were real food-grade system as appropriate antigen delivery systems, and also suitable for the production of ingredients in food industry.
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